IntroductionRecent studies conducted in human/animal cells 1-4 have suggested that homeopathic remedies may have an effect on the gene regulation, initiating a cascade of gene actions to correct the disorder or disease at a cellular level. We have focused our attention on the possiblegene modulation effect of Gelsemium sempervirens , a traditional medicinal plant employed in phytotheraphy and homeopathy as a nervous system relaxant to treat various types of anxiety, pain, headache and other ailments. Previous investigations in our laboratory 5 haveshown a significant anxiolytic-like activity of Gelsemium s . high dilutions in mice emotional models. To follow up on the above evidence, we further investigated the Gelsemium s. mechanism of action in in-vitro neuronal models in order to asses the effects of a wide range ofdrug dilutions on whole genome expression 6-7 . For this gene expression study we used sensitive microarray and real-time PCR techniques, able to survey the whole human transcriptome and specific genes of interest respectively. In our case the high-throughput microarrayanalysis assisted by bioinformatics could provide a strong clue as to the mechanism of action and the biological relevance of ultra-low doses, whereas the real-time PCR was useful in order to validate the microarray data and to further investigate a narrow group of genes, i.e. a panelof human neurotransmitter receptors and regulators, involved in neuronal excitatory signaling. To further investigate the possible mechanisms of gene regulation, we also analysed the possible effect of Gelsemium s. on the methylation status of a group of genes involved in mental disorders.The chief innovation in our experimental design is that it employs a wide range of doses/dilutions: from low dilutions (2c or 3c), where the active substances can still be expected to exert their normal pharmaceutical action, to high dilutions (9c or 30c), where the most controversialprinciples of high dilution pharmacology come into play. We think that this type of investigation may be useful to start building a bridge between the more subjective point of view of symptomatology – basis of the homeopathic remedy choice – and a more accurate and objectiveone, consisting in the identification of specific molecular markers related with the disease and the prescribed remedy.

Experimental setup Global changes in gene expression produced by exposure to high dilutions ofGelsemium s . extracts in human neuroblastoma cells were investigated by and real-timePCR techniques; the methylation analysis was conducted with a PCR Array. Cells were incubated for 24h with the 6 dilutions of Gelsemium s.: 2c, 3c, 4c, 5c, 9c and 30c, produced byBoiron Laboratoires, according to the French Homeopathic Pharmacopoeia. Four replicate experiments were carried out under identical conditions.

ResultsUsing microarray analysis, we observed that the diluted drug Gelsemium s . 2c significantly modulates the expression of 56 genes (49 were downregulated and 7 upregulated) involved in neuronal functions (G-protein coupled receptor signalling pathways, calciumhomeostasis, inflammatory response and neuropeptide receptors). The expression of these genes also decreased significantly, although with small changes, after treatment with medium dilution (3c, 4c and 5c) and high dilutions of Gelsemium s 9c and 30c. In the study conducted using the real-time PCR technique (RT-PCR Array) 7 exposure of a human neurocyte cell-line toGelsemium s . 2c dilution, containing the active principle gelsemine, induced a down-regulation of most genes of this array. In particular, the treated cells showed a statistically significant down-regulation of the prokineticin receptor 2, whose ligand is a neuropeptide involved in nociception (ability to sense pain), and in depression-like behavior. In the latter study, the 9c dilution was notactive. The difference between the two gene-expression studies 6-7is probably due to technical factors: while real-time PCR is the “gold standard” for gene expression analysis of specific genes or small groups of genes,microarray is a powerful screening method for the whole genome and in our conditions exhibited higher sensitivity, detecting extremely low dose effects. In the epigenetic study the Gelsemium s. 2c treatment induced an increased methylation of the homeobox A1 gene (HOXA1), which has a role in neural development and autism spectrum disorders. The mean methylation frequency after treatment with Gelsemium s . 2c was 53% compared to 28% for the controls (p=0.008).The increased level of methylation could indicate a reduced gene expression of this autism related gene.ConclusionsThe results of this study, conducted with three different techniques, provided evidence thatGelsemium s. exerts a prevalently inhibitory effect on a series of genes across a wide dose-range. The results suggest the extreme sensitivity of human gene expression to regulation byultra-low doses and high dilutions/dynamizations of a plant remedy and encourage further efforts in research on this field. Studies using sensitive genetic approaches may be particularly suitable for surveing of the effects of highly diluted natural compounds and for the identification of specific molecular markers related with the disease.AcknowledgementsExperimental work was supported by grants from Boiron Laboratoires to Verona University – Department of Pathology and Diagnostics and from Italian Research Ministry. The authors have no conflicts of interest.